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FADD cleavage by NK cell granzyme M enhances its self-association to facilitate procaspase-8 recruitment for auto-processing leading to caspase cascade.

ABSTRACT: Granzyme M (GzmM), an orphan Gzm, is constitutively and abundantly expressed in innate effector natural killer cells. We previously demonstrated that GzmM induces caspase (casp)-dependent apoptosis and cytochrome c release from mitochondria. We also resolved the crystal structure for GzmM and generated its specific inhibitor. However, how GzmM causes casp activation has not been defined. Here we found that casp-8 is an initiator caspase in GzmM-induced casp cascade, which causes other casp activation and Bid cleavage. GzmM does not directly cleave procaspase-3 and Bid, whose processing is casp dependent. Casp-8 knockdown or deficient cells attenuate or abolish GzmM-induced proteolysis of procaspase-3 and Bid. Extrinsic death receptor pathway adaptor Fas-associated protein with death domain (FADD) contributes to GzmM-induced casp-8 activation. GzmM specifically cleaves FADD after Met 196 to generate truncated FADD (tFADD) that enhances its self-association for oligomerization. The oligomerized tFADD facilitates procaspase-8 recruitment to promote its auto-processing leading to casp activation cascade. FADD-deficient cells abrogate GzmM-induced activation of casp-8 and apoptosis as well as significantly inhibit lymphokine-activated killer cell-mediated cytotoxicity. FADD processing by GzmM can potentiate killing efficacy against tumor cells and intracellular pathogens.

SUBMITTER: Wang S

PROVIDER: S-EPMC3307975 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:UnlabelledEnteropathogenic Escherichia coli (EPEC) has the ability to antagonize host apoptosis during infection through promotion and inhibition of effectors injected by the type III secretion system (T3SS), but the total number of these effectors and the overall functional relationships between these effectors during infection are poorly understood. EspC produced by EPEC cleaves fodrin, paxillin, and focal adhesion kinase (FAK), which are also cleaved by caspases and calpains during apoptosis. Here we show the role of EspC in cell death induced by EPEC. EspC is involved in EPEC-mediated cell death and induces both apoptosis and necrosis in epithelial cells. EspC induces apoptosis through the mitochondrial apoptotic pathway by provoking (i) a decrease in the expression levels of antiapoptotic protein Bcl-2, (ii) translocation of the proapoptotic protein Bax from cytosol to mitochondria, (iii) cytochrome c release from mitochondria to the cytoplasm, (iv) loss of mitochondrial membrane potential, (v) caspase-9 activation, (vi) cleavage of procaspase-3 and (vii) an increase in caspase-3 activity, (viii) PARP proteolysis, and (ix) nuclear fragmentation and an increase in the sub-G1 population. Interestingly, EspC-induced apoptosis was triggered through a dual mechanism involving both independent and dependent functions of its EspC serine protease motif, the direct cleavage of procaspase-3 being dependent on this motif. This is the first report showing a shortcut for induction of apoptosis by the catalytic activity of an EPEC protein. Furthermore, this atypical intrinsic apoptosis appeared to induce necrosis through the activation of calpain and through the increase of intracellular calcium induced by EspC. Our data indicate that EspC plays a relevant role in cell death induced by EPEC.ImportanceEspC, an autotransporter protein with serine protease activity, has cytotoxic effects on epithelial cells during EPEC infection. EspC causes cytotoxicity by cleaving fodrin, a cytoskeletal actin-associated protein, and focal adhesion proteins (i.e., FAK); interestingly, these proteins are also cleaved during apoptosis and necrosis. Here we show that EspC is able to cause cell death, which is characterized by apoptosis: by dissecting the apoptotic pathway and considering that EspC is translocated by an injectisome, we found that EspC induces the mitochondrial apoptotic pathway. Remarkably, EspC activates this pathway by two distinct mechanisms-either by using or not using its serine protease motif. Thus, we show for the first time that this serine protease motif is able to cleave procaspase-3, thereby reaching the terminal stages of caspase cascade activation leading to apoptosis. Furthermore, this overlapped apoptosis appears to potentiate cell death through necrosis, where EspC induces calpain activation and increases intracellular calcium.

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FADD cleavage by NK cell granzyme M enhances its self-association to facilitate procaspase-8 recruitment for auto-processing leading to caspase cascade.

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